Project Summary/Abstract In all chordates, from ascidians to humans, the notochord acts as a fundamental source of support and patterning signals for the developing body plan. In ascidians, the steps required for notochord formation can be clearly visualized because the embryos are translucent and develop very rapidly, and the notochord is composed by only 40 cells. Of note, the last steps of notochord development in ascidians involve the formation of intercellular vacuoles that will eventually coalesce, forming a lumen. Lumen formation, or tubulogenesis, is an evolutionarily conserved process that is reiteratively used by widely different organisms to form tubular structures, and is required during the morphogenesis of a variety of organs. Our laboratory has shown that the transcription factor NFAT5 (Nuclear Factor of Activated T Cells 5) is expressed in the developing Ciona notochord. In vertebrates, NFAT5 is expressed in the centralmost regions of the intervertebral discs, which are formed by notochord-derived cells, and is required to maintain cell homeostasis and osmolarity by activating genes that counteract hypertonic stress. Despite the information available on its role in the survival of notochord-derived cells, the function of NFAT5 in notochord formation is still unknown. We hypothesize that Ciona NFAT5 (Ci-NFAT5) functions in notochord development by controlling the formation of intercellular vacuoles and consequent tubulogenesis. To test this hypothesis, we will determine the identities of genes that are transcriptionally regulated by Ci-NFAT5 (Aim 1). We will also elucidate the mechanisms through which Ci-NFAT5 regulates transcription of notochord genes (Aim 2). Lastly, we will determine the role of Ci-NFAT5 in notochord lumen formation, i.e. tubulogenesis (Aim 3). Completion of these experimental aims will provide the first insight into the role that NFAT5 plays in notochord development, vacuole formation and tubulogenesis. In addition, the proposed work will greatly expand the knowledge of the effectors used by NFAT5 to ensure the integrity of the notochord-derived cells of the intervertebral discs.